Devices and methods for optical detection of nucleic acid hybridization

ABSTRACT

An optical device for determining the presence of a first nucleic acid in a sample comprising a second nucleic acid complementary to the first nucleic acid and able to hybridize with the first nucleic acid under hybridizing conditions, the second nucleic acid being bonded to a solid support, wherein the solid support is formed as a light reflecting surface having a first thickness when bonded to the second nucleic acid, and wherein the light reflecting surface has a second thickness, wherein the first and second nucleic acids are hybridized, and the first and second thicknesses can be distinguished by their effect on the light reflecting properties of said light reflecting surface independent of any label present on the first nucleic acid.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application is a divisional of co-pending application Ser.No. 09/425,072 filed Oct. 22, 1999, which is a continuation ofco-pending application Ser. No. 08/374,151 filed Jan. 17, 1995, which isa Continuation in part of U.S. application Ser. No. 07/965,661, filedSep. 17, 1992, now abandoned, which is a continuation of U.S. Ser. No.07/260,317, filed Oct. 20, 1998, now abandoned, which is a continuationof U.S. Ser. No. 06/832,628, filed Feb. 25, 1986, now abandoned, whichis entitled to foreign priority from West German Application No.P3506703.9-52, filed Feb. 26, 1985, entitled “A Method of SequenceAnalysis of Nucleic Acids in Particular Deoxyribonucleic Acid (DNA) andRibonucleic Acid (RNA), As Well As A Support for Performing the Methodand A Method for Producing the Support,” each of which is herebyincorporated in their entirety herein, including all drawings, tablesand claims.

FIELD OF THE INVENTION

[0002] The present invention relates generally to fields using devicesand methods for detecting nucleic acid hybridization.

BACKGROUND OF THE INVENTION

[0003] The following is a discussion of relevant art, none of which isadmitted to be prior art to the appended claims.

[0004] Nucleic acid probe technology has application in detection ofinfectious disease and genetic and cancer screening. Nucleic acid basedprobe methods offer several advantages over conventional microbiologicalor immunological methods for detection of organisms, as described byNakamura and Bylund (J. Clinical Laboratory Analysis, 6, 73-83, 1992).

[0005] Methods to amplify either the number of copies of the nucleicacid available for detection or the signal generated after hybridizationof the nucleic acid probe have been utilized. A review of nucleic acidbased detection methods and various amplification schemes such aspolymerase chain reaction (PCR), ligase chain reaction (LCR),transcription based amplification, cycling probe reaction, Qβ replicase,and strand displacement amplification may be found in M. J. Wolcott,Clinical Microbiology Reviews, 5, October 1992, pp 370-386.

[0006] U.S. Pat. No. 5,175,270 describes an amplification reagentconsisting of layers of nucleotide polymers containing double strandedand single stranded sections. Each section has an end which is capableof hybridizing with another molecule.

[0007] Probe or hybridization assays are often based on the attachmentof an oligonucleotide probe to a surface in order to capture a targetnucleic acid molecule (analyte) from a sample. The attachment of thisprobe to the surface may be through covalent bonds or through a varietyof passive absorption mechanisms (e.g., hydrophobic or ionicinteractions).

[0008] U.S. Pat. No. 5,279,955 describes an immobilization process whichuses a heterofunctional cross-linker for a plastic support. Thecross-linker consists of a central ring which is hydrophobic andinteracts with the plastic, and a hydrophilic chain which terminates ina group capable of reacting with a nucleic acid. Covalent attachment isachieved through a succinyl-olivetol-N-hydroxysuccinimide.

[0009] U.S. Pat. No. 5,262,297 describes immobilization of a probethrough co-polymers which contain reactive carboxylic acid groups and an8-50 atom spacer with two or more unsaturated groups within the spacer.

[0010] U.S. Pat. No. 5,034,428 describes an immobilization process forprobes in which a monomer is joined onto a hydrophilic solid supportwhich can be irradiated in an oxygen free atmosphere. This methodprovides for covalent attachment of the probe.

[0011] U.S. Pat. No. 4,806,546 also describes an immobilization processfor an amide modified nylon. The method relies on an amidine linkageunder anhydrous conditions in the presence of an alkylating agent.

[0012] Maskos and Southern, 20 Nucleic Acids Research 1679, 1992,describe a linker system for the attachment of a nucleic acid to a glasssupport. The linker system allows for the chemical synthesis of a strandof nucleic acids on the surface. The primary advantage of the linker isthat it is stable to an ammonia treatment which is required in thesynthesis of the polynucleotide. A hexaethylene glycol spacer isincorporated into the linker which attaches to the glass through aglycidoxypropyl silane which terminates in a primary hydroxy group. Thesilane is condensed onto silane groups on a solid support. Additionalcross-linking may be obtained by introducing water so that the epoxidegroup is cleaved to a diol. An acidic solution facilitates this process.The length of the linker may be varied by changing the spacer toethylene glycol, pentaethylene glycol, etc.

[0013] Nucleic acid probes that have hybridized to their target sequenceare detected based on various methods that introduce a detectablechemiluminscent, fluorescent or other identifiable label into a nucleicacid probe. Several of these techniques are described in U.S. Pat. Nos.4,968,602, 4,818,680, 5,104,791, and 5,272,056, and Internationalapplications WO91/00926 and GB2169403A.

[0014] Arnold et al., U.S. Pat. No. 5,283,174 describe the use of achemiluminscent label with DNA probes. The label is composed of anacridinium ester and has a number of desirable properties. It is stableto hybridization conditions, light is emitted only upon exposure to analkaline peroxide, the emission kinetics are rapid, and the label on theunhybridized probe can be destroyed without an impact on the signalgenerated by hybridized probe.

[0015] U.S. Pat. No. 5,089,387 describes a diffraction assay for thedetection of DNA hybridization. In this invention, a solid support,generally silicon or polysilicon, is coated with a DNA probe. Thesesurfaces are required to inherently adhere the DNA probe to the surface.Once the surface is coated with the probe, the surface is selectivelyinactivated to provide a series of very strictly controlled reactiveprobe lines for the generation of the diffraction grating. The unreactedsurface is required to be non-light disturbing. The diffraction gratingis only generated upon the addition of the analyte to the surface. Theangle of diffraction is a function of the wavelength of incident lightand the density and spacing of the individual gratings on the surface. Asingle detector or a multiple detector array may be used to detect andmeasure the light from all desired orders of the diffracted light.

[0016] Mixed phase systems have typically been used to performhybridization assays. In mixed phase assays the hybridizations areperformed on a solid phase such as nylon or nitrocellulose membranes.For example, the assays usually involve loading a membrane with asample, denaturing the DNA or creating single stranded molecules, fixingthe DNA or RNA to the membrane, and saturating the remaining membraneattachment sites with heterologous nucleic acids to prevent the probereagent from adhering to the membrane in a non-specific manner. All ofthese steps must be carried out before performing the actualhybridization.

SUMMARY OF THE INVENTION

[0017] This invention features improved devices, and methods forproducing and using optical devices, for detecting the presence oramount of a specific target nucleic acid within a sample. The currentinvention is based on a probe coated substrate which is opticallyactive. Surfaces can be pre-selected for the type of optical thin filmdetection to be employed, and enable direct detection of thehybridization reaction through the interaction of light with thin films.Detection of specific target nucleic acid sequences is also referred toas sequence analysis.

[0018] This invention also describes materials and methods for producingoptically active solid supports or devices for use in nucleic acidhybridization assays and immobilization of nucleic acid probes to suchsurfaces. These surfaces are compatible with a wide range of opticalthin film detection methods, all of which utilize some interaction ofthin films with light. Such optical thin film detection methods includeoptical interference, ellipsometry—comparison, null, photometric andother modifications, attenuation of polarized light at non-Brewsterangles, profilometry, scanning tunneling microscopy, surface plasmonresonance, evanescent wave techniques, reflectometry, or atomic forcemicroscopy.

[0019] The direct optical thin film detection methods of the currentinvention are extremely sensitive to changes in mass at the surface ofan optically active substrate. These optical thin film detection methodsprovide increased sensitivity for hybridization assays without theintroduction of signal generating labels or pre-assay targetamplification with its accompanying complexity and limitations. Thus,assay protocols utilizing optical thin film detection methods aregreatly simplified, are more rapid, and less costly than conventionalindirect assay methods. Total assay times may vary from one hour to afew minutes from the initiation of the assay protocol (i.e., from thetime that the target nucleic acid containing sample is contacted withthe device). Such devices also allow for assay results to be visualizedas a color change or detected by instrumented formats.

[0020] The methods and devices of the claimed invention by concentratingon improving characteristics of the surface immobilized probe anddetection of the hybridization reaction without the use of a labelledcomponent is allow for rapid, convenient, and sensitive assays fordiagnostic (routine clinical) and research use.

[0021] In a first aspect the invention features an optical assay devicefor detecting the presence or amount of a target nucleic acid. Thedevice includes an optically active substrate capable of producing athin film effect, and exhibiting a first set of reflective andtransmissive properties in response to light impinging thereon, andexhibiting a second set of reflective and transmissive propertiesdifferent from said first set, in response to light when the targetnucleic acid is bound to a target specific capture probe and creates achange in mass on said optically active substrate. The target specificcapture probe is attached to an attachment layer that is present on theoptically active layer. Detection of the second set of reflective ortransmissive properties indicates the presence or amount of the targetnucleic acid.

[0022] An “optically active substrate” comprises an optically activesurface. Such a substrate may consist of more than one layer(multi-layered), for example; base material, a conducting metal layer ofaluminum, chromium, or a transparent conducting oxide, and a layer ofamorphous silicon, wherein the metal layer is positioned adjacent theamorphous silicon. Alternatively, the multi-layered substrate maycomprise a layer of base material (any solid material on which opticallyactive layers may be applied), and a layer of amorphous silicon adjacentthe base material. The base material is selected from any of the groupconsisting of glass, fused silica, plastics, semiconductors, ceramics,and metals, and may be either rigid or flexible. The optically activesurface substrate also serves as a solid support for the nucleic acidtarget probe and any amplification components (if necessary) and must bechemically stable to the application of the attachment layer, thenucleic acid probe, and all assay reagents. The properties of theoptically active substrate are matched to the direct optical thin filmdetection method employed.

[0023] An “optically active surface” is a surface that participates inthe generation of an optical effect such that the light impinging uponthat surface is in some way altered. Such optically active surfaces maybe adapted to respond not only to polychromatic light (e.g., whitelight) but also to monochromatic light (e.g., laser light, which may beinherently polarized). The optically active surface may be designed toreflect or transmit light. The substrate may inherently possess anoptically active surface or the substrate may require an opticallyactive surface be applied to introduce the desired opticalcharacteristics. Thus, the optically active substrate may inherentlypossess the properties required for a given thin film detection method,or a surface may be modified to provide the required optical properties.Materials which are suitable for optically active substrates includemonocrystalline or polycrystalline silicon, glasses, ceramics, metals,amorphous silicon on glass, amorphous silicon on plastic, plastics, andcomposites of these materials.

[0024] By “thin film effect” is meant that light impinging on anoptically active surface or substrate is attenuated or modulated in itsreflective or transmissive properties.

[0025] The first set of reflective or transmissive properties is definedas a combination of wavelengths of light, or a spectral distribution, oran intensity of one or more of wavelengths, or a degree or amount ofelliptical polarization, or an amount of polarization rotation. Thesubstrate also exhibits a second set of reflective or transmissiveproperties which is different from the first set. The second set ofreflective or transmissive properties is exhibited in response to thesame light when the target nucleic acid is present on the surface. Thissecond set of reflective or transmissive properties is due to a changein mass on the optically active substrate. The change from one set ofproperties to another can be measured either by use of an instrument, orby eye. The optical active substrate is selected to be compatible withthe method of optical thin film detection.

[0026] The target nucleic acid is selected so that it specificallyidentifies a single organism or gene. The target sequence is selectedsuch that the stringency of the assay conditions will promote or enhancethe specificity of the assay. The target nucleic acid may be a DNA orRNA (rRNA, tRNA, mRNA, small nuclear RNA's (SNURPS)) molecule eitherintact or a fragment thereof covering a range of molecular weights.Fragments may be generated enzymatically, chemically, or by mechanicalshearing. The target may be free or contained within a larger complex,i.e., complexed with protein(s). The target may be single or doublestranded oligonucleotide and the source may be a bacterium, a virus, ahuman cell, and may be isolated from a culture medium or a biologicalfluid.

[0027] A “target specific capture probe” refers to a synthetic orbiologically produced nucleic acid which by design contains specific,complimentary nucleotide sequences that allow it to hybridize to atarget nucleic acid sequence. The probe may be composed of deoxyribose,ribose, or a combination of these nucleotides (chimera) and may besynthesized chemically, isolated from a biological source, or cloned. Itmay be a linear strand or may contain branch points to increase thedensity of immobilized probe. The probe may provide a single or multiplecopies of the sequence complimentary to the target nucleic acid. Thenucleic acid probe or capture probe is selected to specificallyhybridize a target nucleic acid. When more than one probe is used in ahybridization assay, each probe should recognize a unique sequence wellseparated from each other within the target oligonucleotide.

[0028] Hybridization is the process by which two partially or completelycomplementary strands of nucleic acid are allowed to come together,under predetermined reaction conditions, in an antiparallel fashion toform double stranded nucleic acid with specific and stable hydrogenbonds. The nucleic acid capture probe sequence is selected tospecifically interact with the target molecule or analyte at apre-determined degree of stringency. The probe length is pre-determinedto provide the required specificity at the degree of stringency used inthe assay. Assay conditions are set so that the stringency ofhybridization between the capture probe and the target nucleic acidprovides the required specificity.

[0029] stringency of a particular set of hybridization conditions isdefined by the length and base composition of the probe/target duplex aswell as by the amount and geometry of mispairing between the two nucleicacids. It is governed by such solution parameters as concentration andtype of ionic species, type and concentration of denaturing agents,precipitating agents, and the temperature of hybridization. Asstringency increases, longer probes are preferred for the generation ofstable hybrids. The stringency of the reaction conditions control thespecificity of a hybridization assay. For a complete review ofhybridization assay conditions see “Molecular Cloning—A LaboratoryManual”, second edition, Sambrook, Fritisch, and Maniatis. Also,software has been developed that can more accurately predicthybridization conditions than these traditional methods.

[0030] The capture probe may be modified to promote passive adhesion tothe attachment layer or be chemically reactive with other surfaceimmobilized materials to allow covalent attachment. The modificationsshould be made to the sugar-phosphate backbone to prevent stearicproblems associated with attachment at the bases. A linker may beintroduced into the probe sequence to space the probe from the surfaceand facilitate attachment.

[0031] It is critical that the probe on the surface be attached so thatthe individual monomers are free to hybridize with the target withoutstearic inhibition. This may be particularly relevant when simplehydrophobic interactions are used to immobilize the probe because baseinteractions will be blocked. Covalent methods of attachment caneliminate these considerations and also improve the efficiency andkinetics of hybridization. A determination of the correct position forprobe attachment (horizontal or end) may be made empirically. Inaddition, covalent attachment of the probe should not block additionalbinding sites on the surface. Thus, the probe surface density will notbe reduced. Covalent attachment techniques which modify any or all ofthe probe nucleotide residues must be avoided as they could impactsubsequent hybridization reactions.

[0032] By “mass change” is meant a modification (increase or decrease)in the amount of material at the surface of an optically activesubstrate such that one or more of the reflective and/or transmissiveproperties of the optically active substrate are altered.

[0033] In many cases the optically active substrate selected inconjunction with a specific optical detector is not readily reactive norwill it retain the nucleic acid probe. Thus, the surface must bemodified with a material which enhances or assists in the immobilizationof the probe onto the optical substrate. These materials are referred toas attachment layers. The materials selected for the attachment layermust provide the following characteristics.

[0034] Attachment layers must react either in a covalent or very strongnon-covalent manner with the optical substrate. The probe may be adheredto the attachment layer either covalently or passively. The attachmentlayer may be activated to react with the probe, the probe may beactivated to react with the attachment layer, or both may be chemicallyactivated to react with each other. The attachment layer can be selectedto determine the type of interaction (hydrophobic, hydrophilic, ionic,hydrogen bonded) which occurs between itself and the capture probe.Attachment layers may be applied to the surface of the optical substrateby spin coating, solution coating, vapor deposition, spray coating orthe like. The layer must be applied in a uniform fashion from a solventsystem which will not damage the optical substrate or the chemicalproperties of the attachment layer. Ideally, the solvent will volatilizefrom the surface film during the final curing process.

[0035] In a preferred embodiment the invention features a device with anamplifying probe reagent able to bind to the target nucleic acid andcreate an increase in mass change on the optically active layer, withoutdisrupting the thin film effect, when the target nucleic acid is boundto the target specific capture probe.

[0036] An “amplifying probe reagent” is designed to increase the mass ofthe immobilized target nucleic acid on the optically active substrate.This amplification may be required to increase the sensitivity of thehybridization assay in the optical thin film detection system. Theamplifying probes are attached to another catalytic component orparticle which will increase the mass when the target nucleic acid iscaptured on the surface. The only requirement for such a material isthat it can be tightly associated with the amplifying probe and that theresulting mass deposited at the surface not disrupt the thin filmeffect. When a catalytic component is attached to the signal amplifyingprobe, or amplifying probe, it may act on yet another material, furtherincreasing mass on the optical substrate. There may be a surfaceimmobilized capture probe and one or more signal amplifying probes.

[0037] By “increase in mass” is meant that the mass change produced isgreater than that produced by the binding of the target nucleic acid.

[0038] By “without disrupting the thin film effect” is meant that theoptical effect generated by light impinging on the optically activesubstrate is not destroyed by the introduction of additional materialsto the surface of the substrate. For example, materials which wouldscatter light are disruptive to a thin film interference or otherreflective techniques, as they would artificially reduce the signalavailable to a detector.

[0039] In further preferred embodiments the amplifying probe reagentcomprises a target specific nucleic acid sequence and a catalyticcomponent or a particle; the catalytic component is capable ofinteracting with additional materials to increase the mass change andresult in a second set reflective and transmissive properties withoutdisrupting the thin film effect; the catalytic component is an enzyme;and the particle is selected from the group consisting of metallicparticles, silica particles, and film forming latexes.

[0040] By “catalytic component” is meant any material which can interactwith a substrate to create an insoluble product. Examples of thesematerials include enzymes, metals and other components which cause areaction to occur faster than it would occur in the absence of thatmaterial.

[0041] By “particle” is meant a material such as a film forming latex orsmall non-scattering metal or glass particles.

[0042] By “additional materials” is meant an assay component which isspecifically modified by the catalytic component to produce aprecipitating material. The precipitating agent may be a substrate foran enzyme, e.g., containing 3,3′,5,5′-tetramethylbenzidene when theenzyme conjugate has an immobilized peroxidase or the precipitatingagent is a substrate containing 5-bromo-4-chloro-3-indolyl phosphatewhen the conjugate is alkaline phosphatase.

[0043] The following preferred embodiments are applicable to the devicewith and without the use of an amplifying probe reagent. The attachmentlayer is selected from the group consisting of polymeric siloxanes,mixtures of polymeric siloxanes, film forming latexes, and silylmodified nucleotides.

[0044] The optically active substrate is selected from the groupconsisting of silicon, glass, amorphous silicon, plastic, metals,amorphous silicon on glass, amorphous silicon on plastic, and compositesof these materials.

[0045] An anti-reflective film may be provided between the opticallyactive substrate and the attachment layer. This film which may be formedfrom silicon nitride, silicon oxides, titanium dioxide, siliconoxynitride or cadmium sulfide and the like. This film acts to causeincident light to undergo interference such that a specific color isproduced on the surface of the substrate. This film interacts with otherlayers on the substrate to ensure that a color change or wavelengthintensity change is observed when the target nucleic acid is present onthe device.

[0046] In a preferred embodiment the anti-reflective film is selectedfrom the group consisting of silicon nitride, composites of silicon andsilicon oxides, ti tanium dioxide, titanates, silicon carbide, diamond,and cadmium sulfide.

[0047] In a further preferred embodiment detection is by an optical thinfilm detection method selected from the group consisting ofellipsometry, optical interference, attenuation of polarized light atnon-Brewster angles, profilometry, reflectometry, scanning tunnelingmicroscopy, atomic force microscopy, surface plasmon resonance,evanescent wave techniques, interference spectroscopy, and various otherforms or combinations of polarimetry, reflectometry, spectroscopy, andmicroscopy. This invention concerns the application of such technologiesfor the direct detection or measurement of changes in the thickness,density, refractive index, optical thickness, or mass of thin filmsresulting from the concentration-dependent immobilization of targetnucleic acid on a surface coated with a suitably selected probe. Allthese properties maybe applicable when a thickness or mass changeoccurs. Such thin film assay technologies directly detect or quantitatethe material of interest, and are alternatives to conventional solidphase assays.

[0048] In other preferred embodiments the first and second set ofreflective or transmissive properties are visual interference colors;the first and second set of reflective or transmissive properties arechanges in the degree of rotation observed in polarized light; the firstand second set of reflective or transmissive properties are changes inthe ellipticity of the impinging polarized light.

[0049] By “visual interference colors” is meant the change ininterference colors produced by modifying an anti-reflective layer.

[0050] By “degree of rotation observed in polarized light” is meant theattenuation of the degree or amount of rotation measured in polarizedlight incident upon the optically active substrate before and afterreaction with the target molecule as measured by a change in intensityof the incident light.

[0051] By “changes in ellipticity of the impinging polarized light” ismeant the attenuation of the ellipticity measured in polarized lightincident upon the optically active substrate before and after reactionwith the target molecule as measured by a change in intensity of theincident light.

[0052] In additional preferred embodiments the target nucleic acid isDNA, rRNA, mRNA, tRNA, small nuclear RNA, or complexes of said nucleicacid with other materials; the target nucleic acid is single stranded ordouble stranded; the target nucleic acid is obtained from a bacterium, avirus, a human cell, a culture, serum, plasma, blood, urine, sputum,tissue sample, or other biological fluid. Target nucleic acid isobtained from a source by the use of routine extraction and isolationprocedures familiar to those who practice the art. The target nucleicacid may also require denaturation to allow for hybridization with thecapture probe, such procedures are also standard in the art.

[0053] In a further preferred embodiment, the capture probe is asynthetic or biologically produced nucleic acid which may consist ofDNA, RNA, or a chimera; the capture probe is a linear strand providingsingle or multiple copies of a sequence complimentary to the targetnucleic acid; the capture probe is a branched, multi-copy sequencecomplimentary to the target nucleic acid; the capture probe is adheredto the attachment layer by hydrophobic interactions; the capture probeis adhered to the attachment layer by hydrophilic interactions; thecapture probe is adhered to the attachment layer by ionic interactions;the capture probe is adhered to the attachment layer by hydrogenbonding; the attachment layer comprises a film forming latex and thecapture probe is a chimeric probe which is covalently attached to theattachment layer.

[0054] By “chimera” is meant that the capture probe consists of both DNAand RNA. For example, a single ribonucleotide maybe incorporated into aDNA capture probe and is used in covalent attachment of the captureprobe to the optically active surface. The ribonucleotide residue may betreated with an oxidizing agent such as periodate to produce adialdehyde which is reactive with a carbonyl hydrazide modified surface.A covalent modification of the optically active substrate is produced.

[0055] By “branched multi-copy sequence” is meant a probe with a branchpoint such that from emanating branches a copy of the specific capturesequence is extended, thus presenting multiple copies of the captureprobe.

[0056] In a second embodiment the invention features a method fordetecting the presence or amount of a target nucleic acid. The methodconsists of: preparing a sample, potentially containing a target nucleicacid, for hybridization, contacting the sample with a capture probesupported on an optically active substrate under conditions such thatthe probe specifically hybridizes to the target, and determining theamount or presence of the target on the optically active substrate by anoptical thin film detection method.

[0057] By “preparing a sample” is meant treatment of a sample so that atarget nucleic acid is in a condition that would allow for hybridizationwith a capture probe and includes extraction and isolation proceduresknown to those skilled in the art.

[0058] In a preferred embodiment the target nucleic acid after beingbound to the capture probe is contacted with an amplifying probereagent.

[0059] In a third embodiment, the invention features a method forincreasing capture probe density whereby the probe is applied to anattachment layer on an optically active substrate by spin coating. Thedensity of the immobilized probe is increased by this process relativeto a conventional solution coating process.

[0060] By “capture probe density” is meant the amount of capture probeavailable to bind the target nucleic acid or the number of captureprobes per specific amount of surface area.

[0061] By “spin coating” is meant the standard semiconductor coatingprocedure.

[0062] In a fourth aspect, the invention features a kit for an opticalassay for a target nucleic acid having a test device with an opticallyactive substrate with an attached capture probe which is reactive withthe target nucleic acid, and a reagent adapted to react with the targetbound to the surface to alter the mass on the surface. Preferably, thereagent is an enzyme conjugate or a polymeric film forming latex.

[0063] The kit comprises an optically active substrate coated with atarget specific capture probe. The kit may contain reagents for theextraction of the target oligonucleotide from a biological sample,reagents to destabilize the initial target duplex or other secondarystructure, hybridization reagents, stringency washes, sample processingtubes and transfer pipettes, and amplifying reagents if required.

[0064] In a fifth embodiment the invention features an optical assaydevice for detecting the presence or amount of a target nucleic acidcomprising: an optically active substrate exhibiting a first set ofreflective properties in response to light impinging thereon, andexhibiting a second set of reflective properties different from thefirst set, in response to the light when the target nucleic acid isbound to a target specific capture probe so as to result in a masschange on the optically active substrate. The target specific captureprobe attached to said optically active substrate, and detection of saidsecond set of reflective properties indicates the presence or amount ofsaid target nucleic acid.

[0065] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0066]FIG. 1 is a chart comparing the amount of HRP/TMB precipitation onvarious attachment surfaces coated with biotinylated DNA. Solidrectangles represent DNA and conjugate. Darkly stippled rectanglesrepresent conjugate only. Lightly stippled rectangles represent DNAonly. Striped rectangles represent no DNA and no conjugate. The y axisrepresents thickness in angstroms normalized to the substrate thickness.The x axis represents the various attachment chemistries.

[0067]FIG. 2 is a chart comparing the thickness of alkaline phosphatasereaction product on a T-polymer surface with the hybridizationconcentration of M13mp18 DNA. Solid rectangles represent DNA andconjugate. Darkly stippled rectangles represent conjugate only. Lightlystippled rectangles represent DNA only. Striped rectangles represent noDNA and no conjugate. The y axis represents thickness in angstromsnormalized to the substrate thickness. The x axis representsconcentrations of M13mp18 DNA in the initial hybridization reaction.

[0068]FIG. 3 is a chart comparing the absorbance of substrate at 410 nmwith the composition of spin coating solutions of biotinylated DNAcoated onto T-polymer surfaces which were subjected to various postcoating treatments. Solid rectangles represent no salt in the coatingsolution. Darkly stippled rectangles represent medium salt concentrationin the coating solution. Lightly stippled rectangles represent high saltconcentration in the coating solution. The y axis represents absorbanceat 410 nm. The x axis represents post coating treatment: incubationtimes and temperatures.

[0069]FIG. 4 is a chart comparing the amount of capture probe remainingbound under various stringency conditions. Capture probe was initiallycovalently bound (solid rectangles); adsorbed (stippled rectangles) orrepresent non-covalently bound (striped rectangles). The y axisrepresents absorbance at 450 nm. The x axis represents time ofincubation at 68° C. in water.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] The following descriptions are for further illustrating thevarious embodiments of the present invention and are in not intended tobe limiting in scope.

[0071] The first component required is the optically active substrate.The substrate may be intrinsically optically active or modified toprovide the appropriate properties for the detection method selected.Detection methods described include a visual interference assay andnumerous instrumented systems including ellipsometry-comparison, null,photometric and other modifications, attenuation of polarized light atnon-Brewster angles, profilometry, scanning tunneling microscopy,surface plasmon resonance, evanescent wave techniques, or atomic forcemicroscopy.

[0072] This invention is suited to use of a variety of optically activesubstrate materials and formats based on the needs of the end user. Theoptically active substrate can be formed of, or have coated on it, amaterial that provides either diffuse or specular reflection, it may berigid or flexible, reflective or transmissive. Devices designed forinstrumented analysis do not require an anti-reflective (optical thinfilm) coating on the substrate, while those designed for viewing by eyerequire such a coating. Criteria useful for selecting an opticalsubstrate for instrumented applications, or for viewing by eye of acolor-signal generating application, are presented below.

[0073] A wide range of rigid materials may form the optically activesubstrate, including glass, silicon, fused silica, plastic, ceramic,metal, and semiconductor materials. The substrate may be of anythickness desired. Flexible optical substrates include thin sheets ofplastic and like materials. Most substrates require only a standardsolvent, plasma etching, or acid cleaning, well known to those skilledin the art, before subsequent layers may be deposited on them.

[0074] The surfaces of many solid materials, such as glass, andsemiconductor materials, such as silicon, metals, etc., are sufficientlysmooth to provide specular reflection if they are polished. For use in areflection-based assay the major requirement in selecting an opticalsubstrate is that the reflection occur, or be made to occur, only at theupper surface. This is especially critical for devices which include aninterference film which are viewed by eye. This is accomplished by vapordeposition of a thin metal film on the substrate, and attachment ofsubsequent optical layers by techniques known to those skilled in theart. For example, the uppermost surface of a glass substrate may becoated with a layer (amorphous silicon) to prevent unwanted reflectionsfrom the lower surface.

[0075] If the substrate is to be used in a reflection mode, and ispartially or fully transparent, it may be coated with an opaque materialto block transmitted light and allow reflection to occur only from the,upper surface. For example, a glass substrate may be coated with a layerof aluminum, chromium, or other transparent conducting oxide, bymounting in a vacuum chamber facing an aluminum-filled tungsten boat.The chamber is evacuated to a pressure of 1×10⁻⁵ Torr. Current is passedthrough the tungsten boat, raising it to a temperature at which thealuminum deposits on the substrate at a rate of 20 Å/second for 100seconds, coating the glass with an opaque layer of aluminum having athickness of 2000 Å. Thinner layers of aluminum or chromium may also beused to eliminate any back surface reflections. Non-conductingdeposition techniques may be used to deposit the metal film.

[0076] The aluminum-coated glass, described above, may be consideredoptically passive. Thus, if it is coated with a layer of hydrogenatedamorphous silicon (a-Si:H), the optical characteristics of the substratewill be derived from the a-Si:H alone. The aluminum-coated glass isrequired only when the amorphous silicon deposition process requires aconducting surface. Techniques which do not require the use of aconducting surface for the deposition of amorphous silicon are known. Toproduce this substrate, the aluminum-coated glass is mounted on one oftwo opposing electrodes in plasma-enhanced chemical vapor depositionsystem. The system is evacuated, and the substrates are heated to 250°C. A constant flow of silane (SiH₄) gas into the chamber raises thepressure to 0.5 Torr. A plasma is struck by applying 10 mW/cm² of RFpower to the electrodes. A film of a-Si:H deposits on the substrates,and grows to a thickness of approximately 1000 nm in about 75 minutes.The a-Si:H so formed may form the first optically functional layer onthe test surface.

[0077] A glass substrate coated only with a-Si:H (without the aluminumlayer) is also useful in this invention. Transparent substrates, such asglass, fused silica, sapphire, and many plastics may be used ininstrument transmission measurements, without additional modification.Color-signal generation visible to the eye is possible with atransmissive substrate where the anti-reflection properties of thecoatings are determined from the transmitted light.

[0078] In one example, the optical substrate is formed from a siliconcrystal which is grown and extruded to 4 inches in diameter and thendiamond sawed to form a wafer. The wafers are treated with chemicaletchants to smooth the surface and reduce flaws. The wafers are lappedor ground with aluminum oxide, titanium oxide, or silicon carbideparticles in a talc slurry. The initial grain size is large andsuccessively smaller particle sizes are used to produce an increasinglysmoother surface. Both sides of the wafer are subjected to this process.The final lapping process leaves a very diffusely reflective surface.Wafers may be further processed with chemical or plasma etching tomodify the diffuse reflecting characteristic of the substrate. Once thewafers are lapped, they are cleaned using the following process or aknown modification thereof: the wafers are sonically cleaned with acationic detergent, followed by a rinse with 18 megaohm water. Then theyare cleaned with an anionic detergent, followed by a rinse in 18 megaohmwater. They are ultrasonically cleaned with an aqueous ammonia solutionmade of 370 ml of 30% H₂O₂, 250 ml of aqueous ammonia and 9 gallons ofwater, and are rinsed in a cascade of water with the final rinse beingwith 0.1 micron filtered water. They are then spin-dried and are readyfor optical coating. An alternative to this procedure is the “RCA Clean”described in Polymer Surfaces and Interfaces, edited by W. J. Feast andH. S. Munro, John Wiley and Sons, N.Y., N.Y., page 212, 1987.

[0079] The substrate material may be cut, sawed, scribed, laser scribed,or otherwise manipulated into the desired test piece configuration.Suitable test pieces for a single use assay are 0.5 cm² to 1 cm² with0.75 cm² being preferred. Test piece sizes are not restricted to theabove, as alternative formats may require substantially more or lessreactive test surface.

[0080] For the color-signal generation methods only, substrate selectionwill determine the characteristics of the anti-reflective material ormaterials used in subsequent coating steps. The simplest description ofa single optical thin film is that the substrate is coated with a thinlayer of material such that reflections from the outer surface of thefilm and the outer surface of the substrate cancel each other bydestructive interference. Two requirements exist for exact cancellationof reflected light waves. First, the reflections must be 180° out ofphase and, second, they must be of equal amplitude or intensity.

[0081] In the reflection mode, the optical thin film properties of thecoatings of a device of this invention suppress the reflection of somewavelengths of light and enhance the reflection of others. This causesthe suppressed wavelengths of incident light to enter the substrate, oran opaque coating on the substrate where they are absorbed. Most of thelight of other wavelengths, whose reflection is not suppressed, does notenter the coated substrate and is reflected, however, some componentsmay be absorbed. As the optical thickness of the coating changes, therange of wavelengths in the reflected light changes. In transmissionmode, the properties of the coatings suppress the reflection of somewavelengths of light and enhance the reflection of others, as in thereflection mode. This causes the suppressed wavelengths of the incidentlight to enter the substrate and to be transmitted. Light of otherwavelengths, whose reflection is not suppressed to as great an extent isreflected, and transmitted to a lesser extent. As the optical thicknessof the coating changes, the range of wavelengths in the transmittedlight changes.

[0082] Where visible signal generation is required, the final assayresult may also be measured by instrumentation. Ideally, for theproduction of a perfect interference film using only the a nucleic acidprobe, and an optical substrate, the substrate should have anapproximate refractive index of the square of the refractive index ofthe receptor layer (see below), i.e., (1.5)² or 2.25 (variations in thisnumber can still provide useful devices of this invention). The materialselected should be mechanically stable to subsequent processes,reflective, and of known refractive index. It is not always possible tomatch the optical substrate to a particular film, for example, abiological film. In these cases, an intermediate optical thin film mustbe used to compensate for the lack of a suitable optical substrate. Foreye-visible color-signal generation, the substrate material is subjectto two restrictions: first, it must adhere to the optical thin filmmaterial, and second, in the simplest case, the refractive index of thesubstrate should approximately equal the square of the refractive indexof the material directly above it or, on a more complex test surface,the refractive index of the substrate should be selected to fitgenerally one of the formulae in Table 3, pp 8-48 to 8-49, of the“Handbook of Optics”. For example, use of a silicon wafer with arefractive index of approximately 4.1 allows a test surface to bedesigned with a wide variety of corresponding optical thin films oranti-reflective materials. The material should be coated to a thicknessof a quarterwave for the wavelengths to be attenuated, or variations inthe formulae. Those skilled in the art will realize that various othersubstrate materials are equally suited for use as a test surface if theysatisfy the above criteria.

[0083] The optical thin film coating is deposited onto the surface ofthe substrate by known coating techniques; for example, by sputtering orby vapor phase deposition in a vacuum chamber. Various other usefulcoating techniques are known to those skilled in the art. Materialsuseful as optical thin film coatings are formed of clear material whichis significantly transmissive at the thickness utilized, and suppressessome wavelength of reflective light when coated onto the substrate. Thefilm, once deposited onto the optical substrate, is also stable tosubsequent processes.

[0084] Preferably this test surface will have fewer optical layers, butmore complex test surfaces possessing more layers corresponding to theknown formulae. As already noted, the theoretical calculations are thestarting point for material selection. Theoretical considerations may beused to determine which materials are compatible with a pre-selectedsubstrate. The coating thickness may be set at the predeterminedquarterwave thickness or to a preselected interference color. However,for the construction of a probe/optical thin film composite of thisinvention a number of adjustments are required to the initial opticalthin film coating. An empirical optimization scheme is detailed below.

[0085] A model was developed to select an optimal backgroundinterference color for any particular combination of substrate, opticalthin film (AR film), attachment layer and nucleic acid probe. Since themathematical models developed to date are not effective to provideuseful devices of the present invention, these models are used only as astarting point in the device construction. Optimization is necessary toprovide a device of this invention. For illustration purposes only, theselected substrate was a silicon wafer and the optical material selectedwas silicon nitride. The most highly contrasting colors observed were ayellow-gold changing to magenta with an increase in mass on the testsurface.

[0086] A method for selection of the optimal thicknesses of each layerfor a device of the present invention is disclosed for a silicon nitridefilm on silicon. In step 1, a silicon substrate is provided either witha specular or non-specular surface. A silicon nitride film is providedon this surface in steps 2 and in step 3, this film is eroded away in astepwise fashion by heating and stirring in an appropriate solution. Thetiming of each step is selected such that the portion which is subjectedto erosion for the longest period of time exhibits a pale gold color,while that portion which is not exposed to erosion exhibits a deep bluecolor. In steps 5 and 6 respectively, an attachment layer and areceptive material layer for the target nucleic acid to be detected areprovided on the silicon nitride. These layers are provided in athickness which may be determined empirically, or can be similarlyoptimized (e.g., in this stepwise fashion) if so desired. In step 7, anassay is performed with three portions of the strip being treated in adifferent manner such that a negative response, a weak response, and astrong response can be recorded. The thickness of silicon nitride usefulin the invention can be determined by those sections providing thestrongest weak positive response in the test while maintaining a cleannegative response.

[0087] Specifically, a silicon wafer was prepared with a thick coating(800 Å) of silicon nitride so that the wafer appeared to be a deep blue.Then the optical thin film material was etched off the wafer in a hot,phosphoric acid bath to produce a wedge of interference colors. Theoptical material was etched such that 300 Å remained at one end of thewedge and 700 Å remained at the other end of the wedge. (At 180° C. thesilicon nitride was removed at approximately 20 Å per minute.) Theoptimal film thickness is most readily selected based on the compositetest surface analysis. This process maximizes the visual contrastobtained for the specific assay under development.

[0088] Silicon nitride is easily etched to produce the wedge ofthicknesses needed for this empirical evaluation. Many materials aresusceptible to an acid or base etching processes. Other chemical methodsof etching materials are known. If a desired optical film is not easilyremoved from a particular optical substrate because the film is tooeasily destroyed, or the optical substrate is not stable to the requiredetchant, another method of generating the wedge may be used. Forinstance, monocrystalline silicon is not stable to prolonged exposure tobasic solutions. If an optical film on silicon requires a basic etchantthe wedge can not be generated using a chemical approach.

[0089] Several alternatives exist; first, the optical film may bedeposited on an optical substrate which is introduced stepwise into thecoating chamber over a period of time. Each newly exposed section willreceive a thinner coating than the previously exposed section. Second,the substrate may be masked and the mask removed stepwise over a periodof time. Third, several different coating runs each producing adifferent thickness of optical material may be performed. Fourth, ionmilling may also be used to etch certain materials. For any givenoptical substrate and substitute optical thin film, of the samerefractive index as the original optimized optical thin film, theprocess need not be repeated. Minor thickness adjustments may berequired if the refractive index is not exactly that of the originalmaterial.

[0090] Thus, the formulae established for the coating of optical thinfilms are used as a guideline only for the production of a test surfacesuited to a specific binding assay. For a pre-selected substrate, thesquare root dependence of an optical thin film is used to screenappropriate optical materials. Some deviation from the perfect squareroot dependence is acceptable for this invention. The use of aquarterwave thickness of the optical coating is only an initial guide tocoating thickness. Thickness of the optical thin film must thus beempirically derived in consideration of the nucleic acid capture probe.The composite of the nucleic acid capture probe and the optical thinfilm of this invention does not meet the conditions theoreticallyrequired to produce such a film. Neither the thickness nor therefractive index rules are followed. Surprisingly such deviation fromthese accepted formulae results in a test surface which is verysensitive to mass changes or thickness changes.

[0091] While of less importance, the relative thicknesses of each layer,and not just the optical thin film layer, may be varied as describedabove to optimize the final test device for any particular attachmentlayer and receptive material layer.

[0092] Other optical thin film materials that have a similar refractiveindex include, but are not limited to: tin oxide, zinc oxide, chromiumoxide, barium titanate, cadmium sulfide, manganese oxide, lead sulfide,zinc sulfide, zirconium oxide, nickel oxide, aluminum oxide, boronnitride, magnesium fluoride, iron oxide, silicon oxynitride(Si_(x)O_(y)N_(z)), boron oxide, lithium fluoride, and titanium oxide.

[0093] After substrate selection, an appropriate attachment layer mustbe applied to allow the immobilization of the capture probe. The captureprobe may be immobilized by passive adhesion to the surface or throughcovalent methods. While stearic availability of the nucleic acid basesfor hybridization is one consideration, especially for the planarsubstrates of this invention, another is the density of capture probe onthe surface. An optically active substrate can be derivatized with anumber of different attachment polymer types. The attachment layer canbe used to control the hydrophobicity/hydrophilicity of the surface andthus affect the density and availability of the capture probe. Anattachment layer must be stable under the conditions used to attach thecapture probe to the surface and to subsequent washes. The methods ofapplying a variety of attachment layers to an optical substrate areprovided in the examples below. The composition of the attachment layerwill determine the mode of immobilization for the capture probe.

[0094] In a preferred embodiment, the attachment layer is spin coated oraerosol spray coated in a uniform manner. The various intermediatematerials are coated to the substrate at thicknesses between 5 Å and 500Å (thicker amounts can be employed). The layer can be formed of anymaterial that performs the following functions and has the followingcharacteristics: creates a favorable environment for the capture probe,permits the probe to be bound in active, functional levels (preferablyby a cost-effective method), adheres tightly to the optical substrate,and can be coated uniformly.

[0095] Ideally, the surface activation technique should provide acovalent modification of the surface for stability while introducing avery dense uniform or conformal film on the surface of the substrate. Astrongly adsorbed conformal film without covalent attachment may beadequate. Substrates such as monocrystalline silicon, macroscopicallyplanar, uniform optical glasses, metalized glass and plastic, whether ornot coated with an optical layer (i.e., SiO, SiO₂, Si_(x)N_(y), etc.)have a deficiency of available reactive groups for covalent attachment,but are still useful in this invention. Once applied, the attachmentlayer should provide an environment which supports the adherence of aprobe layer by covalent or adsorptive interactions, that is dense andfunctional. This attachment layer must be of sufficient thickness toseparate the capture probe layer from any toxic effects of the initialoptical substrate.

[0096] A general method for covalent surface attachment of DNA captureprobes has been devised. The capture probe is attached through the 3′end. A chimeric capture probe is designed which includes at least asingle ribonucleotide at the 3′ end of the deoxyribonucleic acidpolymer. For evaluation purposes, the 5′ end of the probe is synthesizedwith a biotinylated residue. In an actual assay the 5′ end would not bemodified. The ribose sugar ring in the chimeric probe will open to forma dialdehyde in the presence of sodium periodate by cleaving the 2′-3′bond of the sugar residue (Methods in Enzymology, Volume LIX, 1979, pp.172-181, F. Hansske, and F. Cramer). This opened structure may bereacted with a carbonyl hydrazide modified surface to form a condensedsix membered ring structure by the interaction of the aldehydes with thesurface nitrogen by beta elimination of the 1′ and 4′ hydrogens.

[0097] The capture probe could also be synthesized containing a PsoralenC2 Phosphoramidite which was originally designed to introduce afluorescent label into a nucleic acid probe. This molecule whenphotoactivated will cross-link to any available thymine. The psoralenmodification would be used to attach the capture probe to the surface.It may be desirable to add a linker between the probe and the Psoralenmonomer. It is also important that the probe not contain a TT sequenceor they will dimerize upon photoactivation. Probes can be designed withbranching spacers using phosphoramidite chemistries including5′-Branched modifier C3 or C7 and 3′ Amino-modifier C3CPG to introducemultiple copies of probe to linkers with single points of attachment. Aprimary amine modified spacer could be incorporated into the captureprobe which could then be reacted with an aldehyde coated surface. Imineformation can be promoted by NHS/carbodiimide chemistries and then maybe reduced with NaBH₄. If the oxygen in the phosphate groups of the DNAbackbone is replaced with a sulfur group, then coordination complexeswith metal surfaces can be used to immobilize the probe.

[0098] One of the best approaches for the generation of a longoligonucleotide on the surface is to immobilize an oligonucleotide onthe surface then ligate the remaining sequence onto the surface using aligase reaction. This could be used to covalently attach a probe whichcontains a large number of repeats of the desired sequence. This wouldincrease the efficiency of the capture of the target by improving thestearic availability of the probe for hybridization.

[0099] A hydrophobic surface may be used to immobilize a capture probewhich is modified with a poly T tail. A poly T tail is very hydrophobicand can be used to associate with the surface and drive the attachmentof the probe to the surface. When this approach is used, samples shouldbe treated to remove poly A tails which would associate with the surfaceand block the desired hybridization reaction. Alternatively a poly Tprobe could be used to isolate an mRNA and then specifically identifythe mRNA through an amplifying probe.

[0100] A carbonyl hydrazide surface may be generated by applying a thinfilm of a carboxylate modified polymer (such as a film forming latex) tothe surface by aerosol, spin coating, dip coating, etc. The carboxylateis then treated with N-hydroxysuccinimide and a carbodiimide followed byhydrazine to create the hydrazide surface. It is also possible to use anamine modified surface for direct interaction with the aldehyde groups.

[0101] A capture probe may be modified with a free thiol group at the 3′or 5′ end. This free thiol may be reacted with a surface thiol to from adisulfide bond and thus link the probe to the surface. A mercaptosiloxane may be used to introduce the thiol onto the surface through aspin coating process. Or a polymer may be combined with a siloxane suchas the T-Polymer to create a hydrophobic surface which helps draw theprobe to the surface prior to actual attachment. In this case unreactedsurface thiols must be blocked prior to subsequent processing, andconditions for the assay must not reduce the dithiol to free thiolgroups.

[0102] A capture probe may also be linked to a material which is easilyadhered non-covalently to the surface. When a surface is coated with theT-polymer, proteins strongly adhere to the solid support. Theoligonucleotide is modified at the 5′ end with hexanolamine which can beadded directly to the probe by a nucleic acid synthesizer. A maleimideis synthesized by the addition of 3-maleimidobenzoic acid in basicsolution and purification by anion exchange chromatography. Thismodified capture probe is then incubated with the protein either insolution or coated onto a solid support to couple the probe and protein.The ratio of the probe: protein and other parameters can be set tocontrol the level of probe incorporation and thus the surface densityobtained.

[0103] The capture probe must be tightly associated with the surface soas to minimize the rate of removal, especially during the finalstringency wash processes. Covalent bonds may be susceptible to heat,changes in ionic strength, and reducing conditions. Non-covalenttechniques are susceptible to these conditions and also to the presenceof surfactants or other materials which will compete with the mode ofattachment used. The bond between the surface and the capture probe mustbe stronger than the bond between the nucleic acid hybrids to ensurethat a high stringency wash does not remove the probe from the solidsupport.

[0104] It was discovered that some of the materials that bound captureprobe most effectively had very little in common; some were veryhydrophobic while others were strongly charged. Thus, it is possible totailor the surface to exploit the amphiphilic nature of the probe andthe specific assay application being designed. One type of surface maybe well suited to applications were the sample is extensively processedand is primarily a buffered matrix. Another may be best suited toapplications where the sample matrix is more closely related to theoriginal biological medium.

[0105] Additional materials that could be applied to an opticalsubstrate and serve as attachment layers include hydrophobicisobutyltrimethoxysilane, charged N-trimethoxysilylpropyltri-N-butylammonium bromide, composites of these and previously describedmaterials, and crosslinking reagents such as tetramethoxysilane,polystyrene. One interesting silane N-6,9,-bis (trimethylsilyl)adeninecould be used to introduce an adenine to the surface for synthesis ofthe capture probe on the surface or ligation of a capture probeenzymatically to the surface.

[0106] In some cases a signal amplifying probe or probes may be requiredto achieve the sensitivity desired for the detection of the targetnucleic acid. As with the capture or immobilized probe, the amplifyingprobe may be a DNA or RNA probe. The amplifying probe may be modified toinclude additional mass generating materials. And the oligonucleotidereporter sequence selected should be well separated from the sequencehybridized by the capture probe.

[0107] One possible method of signal amplification is to attach theamplifying probe to horseradish peroxidase (HRP) or alkaline phosphatase(AP) and use in combination with a precipitating substrate. See Renz,M., and Kurz, K., “A Colorimetric Method For DNA Hybridization”, Nuc.Acids Res., 12, 3435-3444, 1984 for a method to couple anoligonucleotide to these enzymes. Catalytic, but non-enzymatic,reactions which have a specific catalyst and reactant and lead to theformation of an insoluble product may also be utilized. Some mechanismmust exist which will allow the attachment of the amplifying probe tothe catalyst.

[0108] Signal amplification reagents can be attached to the nucleicacids by a variety of linking agents including glutaraldehyde, N,N′-o-phenylenedimaleimide,N,N′-oxydimethylenedimaleimide,N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,N-succinimidyl-m-maleimidobenzoate, andN-succinimidyl-3-(2-pyridyldithio)propionate. Signal amplificationreagents can include enzymes, metal particles, latex particles, multiplecopies of an enzyme or multiple enzymes, highly branched nucleic acidsor other similar compounds which can be specifically attached to asecondary probe. These reagents should be selected to specificallyenhance the mass of the immobilized target while not disrupting the thinfilm effect. While the packing of the molecules in the layer do notapproach a true thin film, due to the percent occupancy of all availablebinding sites, they must not disrupt the thin film effect in order tomaximize the quality of the generated signal. Thus, amplificationreagents cannot behave as discrete particles on the surface of theoptical substrate.

[0109] After the sample is contacted with the surface of a test device,an instrument can be used to detect analyte binding. One such instrumentis the Sagax Ellipsometer (see, U.S. Pat. Nos. 4,332,476, 4,655,595, and4,647,207 which disclosures are incorporated in full herein and made apart hereof). Alternate instruments suited to this technology includetraditional null ellipsometers, thin film analyzers, profilometers,polarimeter, etc. If the interference film is included in the testsurface construction, then a simple reflectometer is adequate forquantitation. Unlike conventional ellipsometry, the ComparisonEllipsometer is designed to allow broad field measurements. This featureallows simultaneous measurement of the entire reaction zone. Therefore,measurement errors do not arise because of non-homogeneous binding orreaction patterns.

[0110] For the analysis of specific binding reactions on a test surface,a number of modifications greatly improve the performance of theComparison Ellipsometer. The original design relied on the observer'seye for inspection of the surface.

[0111] A detector may be mounted where the eyepiece is located in theoriginal instrument. It may also be mounted at 90° to the side of thelight path by incorporation of a partially silvered mirror orbeamsplitter set at 45° to reflect a portion of light to a detector, andthe rest to the eyepiece for visual alignment of samples. If the mirroris inserted into the optical path, the spot intensity reaching thedetector will be only a fraction of the light available. If the detectoris directly in the optical pathway without a mirror, 100% of the sampleintensity reaches the detector. When a beamsplitter and eyepiece areincluded in the apparatus, if care is not taken, stray light can beintroduced which degrades the optical signal incident on the detector.

[0112] A photodiode array may be programmed to dedicate individualphotodiodes to measure the intensity of reaction zones or spots, whileother photodiode arrays measure the background, or control zones.Simultaneous measurement of the spot intensity and the backgroundintensity allows each reading to be accurately corrected for testsurface background.

[0113] Either a linear array or a matrix array may be used. A lineararray may only measure along one, pre-set axis of the sample spotdepending on the size and resolution available in the arrays. The matrixarray could measure the entire reacted spot plus background.

[0114] The instrument may also be modified to include a variablemagnification function or a zoom to allow different spots to fill thephotodiode without capturing any background signal. A semi-reflectivemirror was introduced between the zoom and the ocular at 45°. Within theocular, suitably positioned in the middle of the field and in focus wasset a reticle of an ellipse. The reticle was selected to match anaverage sample spot size. On the optical path center line, reflected 90°from the principal axis, was set a mask which matches the size of thereticle. The distance from the center of the mirror to the reticle isthe same as from the center of the mirror to the mask. The mirror wasmounted by adjusting screws so that the image seen within the reticlewould be identical to the image appearing within the mask. Behind themask, a distance of a few millimeters, was mounted a photosensitive cellarranged to only read the light which passes through the mask andtherefore from the selected image. The semi-reflective mirror is of athickness such that a secondary image appears from the second surface.This is eliminated by using a suitably coated thin mylar membrane as thebeamsplitter.

[0115] A constant light source, white light or monochromatic, isprovided by using a power supply that has feedback capabilities. Aphotoresistor is mounted inside the original instrument's lamphouse/heat sink which monitors the light output of the lamp. If thelight output changes a corresponding resistance change occurs, therebyaffecting the current/voltage sent to the lamp.

[0116] The power supply is set to deliver +15 V_(DC) to the lamp whilethe photoresistor is disconnected. When the photoresistor is connected,it maintains the light output at the level that is produced with a +15Vsource. A constant light source is required if the instrument is to beused for quantitation. The instrument may also be modified with a BNCport that will enable the output of the photodiode detector amplifier tooutput to an A/D converter board in a computer or other dedicateddevice. The dedicated device or computer reads the input signal,designates/names and stores the input, manipulates the named input,i.e., conducts statistical analyses, etc., and prints the input data andany other desired calculations derived from the input.

[0117] The reflectometer is a very simple instrument which allowsmeasurement of a color change or a change in intensity. A standardhalogen light source is used. This will provide polychromatic light. Thelight source is positioned relative to the test surface such that themaximum intensity of the incident light impinges the test surface. Thedetector may be a photomultiplier or the like. The angle with which thelight impinges the test surface determines the angle at which thedetector is placed relative to that surface.

[0118] Another useful instrument is a thin film analyzer which uses amonochromatic light source. If the light is not sufficiently linearlypolarized, then a polarizer near the source may be used to polarize thelight. The polarizer must be positioned to allow the maximum intensityof light to pass through to the test surface. By off-setting the initialpolarizer a component of light polarized perpendicular to the plane ofincidence, in addition to the light polarized parallel to the plane, isallowed to interact with the surface. Light impinges the test surface atan angle which is sufficiently removed from Brewster's angle, between 50and 75 degrees off the normal. The polarizer/detector is set at the sameangle from the normal as the incident light source relative to the testsurface. The polarizer is set from 2° to 15° above the setting whichaligns the polarizers for total extinction of light. Incident angles of30° to 40° off the normal provide adequate resolution of very dilutesamples, but may not provide sufficient range for all applications. Thesecond polarizer, or analyzer polarizer, cannot adequately minimize thebackground signal when the light is incident on the surface at anglesgreater than 65°. However, the dynamic range is sufficient to allow forelectronic reduction in the background signal. The light is reflectedfrom the test surface through the polarizer/analyzer combination priorto being measured at the detector. The detector may be a singlephotodiode or a photodiode array. A blank test surface is placed in thesample position and used to align the second polarizer. The secondpolarizer should be positioned at an angle with respect to the firstpolarizer such that it is a few degrees off the minimum (maximumextinction of light through to the detector). Thus, the background ofthe test surface produces a low detectable signal, but the change inlight intensity is now a function of the change in thickness.

[0119] The thin analyzer eliminates the reference surface requirementsof the previous instrument and is easier to reduce in size. Thecomparison based instruments require that a specific reference surfacebe designed for each type of test surface to be used. This limits therange of optical substrates and optical thin films which are compatiblewith a given instrument, unless means for changing the reference surfaceis provided. This new instrument easily accommodates any combination ofthin film and optical substrate using a simple adjustment of theanalyzer. The instrument may provide better thickness resolution. Thisinstrument and the modified Comparison Ellipsometer may be powered witha 9V battery or other rechargeable power supply. This prototype suppliesan increase in numerical aperture, image brightness and focus. Thisallows a much higher level of magnification to be used which isimportant for work with smaller spot sizes. Samples may also be appliedmuch closer to one another than is possible with the ComparisonEllipsometer.

EXAMPLES Example 1

[0120] Evaluation of Attachment Chemistries

[0121] A 5′-biotinylated oligonucleotide (capture probe) was used toevaluate attachment chemistries for the immobilization of DNA captureprobes to optically active surfaces. Several attachment or surfacechemistries are presented.

[0122] Surface 1: T-Polymer

[0123] A 128+/−2 Å layer of T-Polymer-Aminoalkyl T-structure branchpoint polydimethyl siloxane (Petrarch; Bristol, Pa.) was used. A 1:300(v/v) dilution of the T-Polymer was prepared in 2-methyl-2-butanol. Theattachment layer was applied to the silicon wafer by a spin coatingmethod and was cured for 24 hours at 140° C. prior to use. For spincoating, a 300 μl sample of this solution was placed on a 100 mm virgintest silicon wafer by micropipette, although automated aerosol or spraydelivery systems are equally useful, while the wafer was spinning at7,000 rpm on a photoresist spin-coater.

[0124] Surface 2: R-Polymer

[0125] A 70+/−2 Å layer of spin on glass was coated onto a virginsilicon wafer as follows. A solution containing 1.6%, each, of trimethylQAC and octadecyl QAC (quartnery ammonium compound, Petrarch), 36.8%water, 10% methanol, and 50% isopropanol was prepared. A 400 μl sampleof this solution was applied to the wafer which was attached to a spincoating apparatus and spinning at 3000 rpm. The wafer is spun until auniform thin film is generated. The glass is then annealed or cured ontothe wafer by incubating for 2 hours at 155° C.

[0126] Surface 3: Si_(x)N_(y)

[0127] A 499+/−2 Å layer of silicon nitride was applied to the virginsilicon wafer using standard vapor deposition processes known in thesemi-conductor industry.

[0128] Surface 4: RT II

[0129] A 1:1 mixture of the T-Polymer solution with the R-polymersolution was prepared. A 400 μl sample of this mixture was applied tothe wafer which was spinning at 3000 rpm. The wafer was spun until auniform thin film was generated. The polymer layer is cured onto thewafer by incubating for 2 hours at 155° C.

[0130] DNA capture probe was coated onto these wafer surfaces from asolution containing 50 mM sodium citrate, pH 6.0, 0.1 mg/ml carrier DNA,sheared herring sperm DNA, and 600 μM biotinylated DNA, 26-mer. Theprobe sequence was 5′-CGCTAATATCAGAGAGATAACCCAC-3′. Wafers wereincubated in this solution overnight at 4° C. Wafers were removed fromthe solution and washed with 1×phosphate buffered saline containing 0.2%Tween 20™ detergent (PBS/Tween). The wafers were then coated in a BSA(bovine serum albumin) solution for 3 hours at 65° C. The wafers werethen rinsed with PBS/Tween detergent.

[0131] To measure the amount of biotinylated DNA adsorbed to thesurface, the wafers were incubated for 30 minutes with a solutioncontaining streptavidin conjugated to horseradish peroxidase (ImmunologyProducts) was diluted 1:250 in 50 mM MOPS, pH 7.0, containing 3%alkaline treated casein, 0.2% TWEEN20 detergent, and 0.5% Proclin 300(an anti-bacterial agent). Wafers were then washed with deionized waterand dried under a stream of nitrogen. A drop of TMB precipitatingsubstrate was applied to the surface and the wafers incubated for 30minutes at room temperature. Thickness increases (in Angstroms) due tosubstrate deposition were measured using an absolute ellipsometer(Gaertner) which was normalized to the initial substrate thickness. SeeFIG. 1. These experiments were repeated using alkaline phosphataseconjugated to streptavidin and BCIP/nitroblue tetrazolium substratepair. The R-polymer surface (most polar surface used) and the T-polymersurface (least polar surface used) all performed well. These experimentsdemonstrate that DNA can be successfully immobilized to the surface ofthe silicon wafer.

Example 2

[0132] Sensitivity Evaluation in an Un-optimized Assay

[0133] Wafers coated with T-polymer (see Example 1) were coated for 56hours at 4° C. in a solution containing 50 mM sodium citrate buffer, pH6.0, 5×SSC, and 20 μg/ml of the ssDNA capture probe complimentary toM13mp18. The probe sequence was CGCTAATATCAGAGAGATAACCCAC. Probe coatedwafers were removed from coating solution and placed into a blockingsolution containing 5×Denhardt's solution, 0.5% SDS, 1 mg/ml carrierDNA, and 25 mM buffer at pH 6.5. They were incubated 16-18 hours at 4°C. and then rinsed with phosphate buffered saline containing 0.0005%TWEEN20 detergent at pH 7.4. Capture probe coated wafers were hybridizedwith M13mp18 plasmid overnight at 60° C. in a solution containing1×Denhardt's solution, 0.5% SDS, 25 mM MES, pH 6.5, 0.2 mg/ml carrierDNA, 5×SSC, a final concentration of M13mp18 was 500 ng/ml, 1 ng/ml or100 pg/ml. The final hybridization step occurred under the same solutionand incubation conditions as the previous step with a final biotinylatedamplifying probe concentration of 92 μM. The amplifying probe containsstrand sequence from 6249 to 6273 and was biotinylated at residue 6261.The sequence is GCAGGTCGACTGTAGCAGGATGCCGG. All appropriate controlswere performed. Wafers were incubated with a streptavidin alkalinephosphatase conjugate. Precipitating substrate, BCIP/nitrobluetetrazolium, was used to generate an increase in thickness at thesurface of the wafer. Thickness increases were measured using anabsolute ellipsometer (Gaertner). Results for the experiment are shownin FIG. 2. From this experiment, it was concluded that a sensitivity of1 ng/ml and potentially as low as 100 pg/ml was achieved. Thistranslates to a copy number of roughly 10¹⁰ for a very un-optimizedassay.

Example 3

[0134] Spin Coating of the DNA Capture Probe

[0135] Spin coating of the DNA capture probe (M13mp18 fragment6249-6273) was carried out on two wafer polymer preparations: T-polymerand R-polymer. The wafers were prepared as previously described inExample 1. The DNA capture probe was applied using 400 μl of solutionand a speed of 4000 rpm. Three coating solutions were prepared asfollows:

[0136] High Salt Solution:

[0137] 5 μl of 10 mg/ml sheared herring sperm DNA

[0138] 25 μl of 0.50 M Pipes pH 7.0 buffer

[0139] 105 μl of methanol (anhydrous)

[0140] 315 μl of biotinylated DNA stock (316 μg/ml)

[0141] 50 μl 20×SSC

[0142] Medium Salt Solution:

[0143] 5 μl of 10 mg/ml sheared herring sperm DNA

[0144] 25 μl of 0.50 M Pipes pH 7.0 buffer

[0145] 105 μl of methanol (anhydrous)

[0146] 315 μl of biotinylated DNA stock (316 μg/ml)

[0147] 25 μl 20×SSC

[0148] 25 μl H₂O

[0149] No Salt Solution:

[0150] 5 μl of 10 mg/ml sheared herring sperm DNA

[0151] 25 μl of 0.50 M Pipes pH 7.0 buffer

[0152] 105 μl of methanol (anhydrous)

[0153] 315 μl of biotinylated DNA stock (316 μg/ml)

[0154] 50 μl H₂O

[0155] After spin coating, the wafers were broken into 6 pieces (chips)and sorted to undergo the following post coating treatments.

[0156] #1: 60° C., 2 hours

[0157] #2: 45° C., 2 hours

[0158] #3: Room temperature, 2 hours

[0159] #4: 40° C., 4 hours

[0160] #5: 60° C., 4 hours

[0161] #6: Room temperature, 4 hours

[0162] After the post coating treatment process individual 0.75 cm² testsurfaces were cut and placed into assay devices. After the wafers wereplaced in the device they were washed with wash buffer (2×SSC, 0.1%(w/v) SDS) to remove any unbound DNA capture probe. A control wafer foreach surface was included. The control did not include a conjugateincubation step and received substrate only. Test wafers were incubatedfor 15 minutes at room temperature with a 100 μl spot of a 1:1000dilution of a streptavidin/HRP conjugate. The conjugate was aspiratedfrom the surface and then the surface thoroughly washed with washbuffer. A 100 μl spot of the 2 part Kirkegaard and Perry TMB substratewas applied and incubated at room temperature for 10 minutes. Thesubstrate was quantitatively transferred to a microtiter well containing100 μl of 2.5M sulfuric acid and then the absorbance measured at 410 nm.Results using the T-polymer are shown in FIG. 3. Solution coating of theDNA capture probe onto the T-Polymer surface provided a functionalsurface, but only captured approximately ¼ of the probe that the spincoating technique provided. For the solution coating of probe to thesurface, the salt concentration of the coating solution was the mostcritical variable. The optimal coating solution was determined to be0.25 mg/ml of carrier DNA, 6×SSC pH 7.5, room temperature incubation,and the best surface was the T-Polymer.

Example 4

[0163] Adherence of Covalently Bound DNA Under Stringent Conditions

[0164] Virgin silicon wafers were coated with a layer of film forminglatex consisting of free carboxylic acid groups. A 30% stock solution ofTC7A (Seradyn, Indianapolis, Ind.) was diluted to a 0.5% solid inmethanol. A 300 microliter sample was applied to the substrate using thespin coating technique and was cured at 37° C. for 120 minutes prior touse. A final thickness of this material is preferred to be 240 Å. Thesecarboxylic acid groups were reacted with NHS (20 mM),N,N′-dicyclohexylcarbodiimide (DCC, 20 mM) in dioxane. These componentswere mixed on ice for 15 minutes and then incubated for 4-5 hours atroom temperature. Then 32 mmoles of hydrazine was added to leave areactive hydrazide on the surface. The mixture was incubated for 1 houron ice and then overnight at room temperature. A DNA capture probe witha 3′ terminal ribonucleotide residue was treated with sodium periodateat a DNA:NaIO₄ ratio of 1:15 for 1 hour to leave a dialdehyde group onthe polynucleotide. The dialdehyde then adds to the carbonyl surface toform a covalent adduct of the capture probe on to the surface.

[0165] An 18 mer DNA/RNA chimera was utilized as the capture probe. Theprobe was biotinylated at the 5′ end and has a ribonucleotide cytosineon the 3′ end. The DNA sequence was 5′-CGAAGCTTGGATCCGCC-3′ (ribose).The covalently attached capture probe was treated with S1 nuclease todegrade the entire probe from the surface. The S1 nuclease was mixed ina solution of 0.2 mM NaCl, 0.05 M sodium acetate pH 5.4, 1 mM ZnSO₄, and0.5% glycerol to a final concentration of 2 units/ml. A section of thewafer was submerged into 7 ml of the enzyme solution and incubated for10 minutes at 37° C. Wafers were rinsed prior to enzyme treatment inwater for 2 hours at 45° C. The enzyme solution was decanted into testtubes and a small volume of water used to rinse the wafers. The combinedsolution was dried with a SpeedVac system. The pellet was extracted intoacetone and the solution dried. These pellets were re-suspended in 70 μlof water and the A₂₆₀ measured in a microcuvette. The surface density ofthe probe was determined to be 50 ng/cm². Control surfaces where noNaIO₄ was used, no covalent attachment, did not generate signal. Controlsurfaces without DNA or without S1 nuclease gave no signal.

[0166] Wafers with covalently attached capture DNA probe were incubatedat 68° C. in pure water for 4, 7, or 24 hours in order to simulate veryextreme conditions of stringency. Wafers were removed, dried andincubated with a streptavidin HRP conjugate. Unbound conjugate wasrinsed from the surface. The Kirkegaard and Perry two component TMBsubstrate was applied for 20 minutes at room temperature. After fourhours, the intensity of the ELISA was not changed, indicating that thecapture DNA probe on the surface had not been stripped to anysignificant extent. A wafer with adsorbed, non-covalently bound DNAprobe was shown to lose all of its DNA probe under these conditions. SeeFIG. 4.

Example 5

[0167] Estimated Sensitivity Based on Bound Capture Probe

[0168] An 8-mer was synthesized and was 5′ biotinylated and had a 3′ribose. The DNA sequence was 5′-AAAGATGTA ribose)-3′. The 8-mer wasimmobilized using the 15:1 periodate:probe ratio to a TC7 coated opticalsubstrate as described in Example 4. Chips were coated at theconcentrations listed in the table below. The amount of immobilizedbiotinylated DNA was measured by reacting a pre-determined surface areawith a sufficient volume of 1:1000 dilution of astreptavidin/horseradish peroxidase conjugate to cover the test surface.The conjugate and surface were incubated at room temperature for 10minutes. The chips were rinsed 2 times with wash buffer and then water.The Kirkegaard and Perry two component TMB substrate was applied for 20minutes at room temperature. A volume of the substrate was placed in amicrotiter well with 50 ul of 2.5 N sulfuric acid and 50 ul of water.The absorbance at 450 nm was measured in a microtiter plate reader. Theminimum detectable amount of biotinylated DNA was correlated to the copynumber of capture probe used in the initial coating solution. Theresults are shown below. CONCENTRATION OF COATING SOL. COPY # Ave A₄₅₀22 μM 10¹⁵ 0.907 0.22 μM 10¹³ 0.808 0.22 nM 10¹⁰ 0.176 22 fM 10⁶ 0.175 0 0 0.017

[0169] Assuming a one to one correspondence of binding between captureprobe and target sequence, the copy number should represent anestimation of the sensitivity of an unoptimized assay for the detectionof target sequence.

[0170] Other embodiments are within the following claims.

1 4 1 25 DNA Bacteriophage M13mp18 Capture probe complimentary toM13mp18 1 cgctaatatc agagagataa cccac 25 2 26 DNA Bacteriophage M13mp18Amplifying probe complimentary to M13mp18 2 gcaggtcgac tgtagcagga tgccgg26 3 17 DNA Bacteriophage M13mp18 17-mer DNA/RNA chimera 3 cgaagcttggatccgcc 17 4 9 DNA Bacteriophage M13mp18 9-mer 4 aaagatgta 9

What is claimed is:
 20. A support for use in detecting the presence of atarget nucleic acid comprising an optically smooth, flatlight-reflecting surface, said surface having a nucleic acidcomplementary to said target nucleic acid bound thereto.
 21. The supportaccording to claim 20 wherein said nucleic acid bound to said surface isbound by covalent bonding.
 22. The support according to claim 20comprises silicon or glass.
 23. The support according to claim 20,wherein said light reflecting surface comprises a layer of aluminum orsilicon.
 24. The support according to claim 23, wherein said layer ofaluminum or silicon is a layer of a compound selected from the groupconsisting of silicon dioxide, silicon monoxide, and aluminum oxide. 25.The support according to claim 24, wherein said support furthercomprises an anti-reflection layer.
 26. The support according to claim20, wherein said nucleic acid bound to said surface is indirectly boundthrough an intermediate molecule bound to said surface.
 27. The supportaccording to any one of claims 20-26, wherein said support furthercomprises said target nucleic acid bound to said complementary nucleicacid, wherein reflectance from said light-reflecting surface is alteredin comparison to reflectance by said light-reflecting surface in theabsence of said bound target nucleic acid.